Isolation of nucleoli from Ehrlich ascites tumor cells and dynamics of nascent RNA within isolated nucleoli.
Here we describe a new, rapid method for isolating nucleoli from Ehrlich tumor cells that preserves their morphological integrity and high transcriptional activity. Until now, methods for isolation of nucleoli were generally assumed to empty one of their three main compartments, the fibrillar center, of its contents. This new method consists of sonicating cells in an isotonic medium containing MgSO(4), spermidine, and spermine, followed by separation of nucleoli through a Percoll density gradient. Using the nonisotopic approach of labelling with BrUTP, we have further investigated the dynamics of nascent ribosomal RNAs (rRNAs) within morphologically intact isolated nucleoli at the electron microscope level. We show that ribosomal transcripts are elongated in the cortex of the fibrillar center and then enter the surrounding dense fibrillar component.
Thiry
Marc
M
Laboratory of Cell and Tissue Biology, Department of Life Sciences, University of Liège, Liège, Belgium.
Ploton
Dominique
D
D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000378:metabolism*; D002466:Cell Nucleolus / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D008854:Microscopy, Electron / Q000379:methods; D008967:Molecular Biology / Q000379:methods; D012313:RNA / Q000378:metabolism*; D012322:RNA Precursors / Q000378:metabolism; D012335:RNA, Ribosomal / Q000378:metabolism; D014158:Transcription, Genetic; D014544:Uridine Triphosphate / Q000378:metabolism
D012322:RNA Precursors; D012335:RNA, Ribosomal; D012313:RNA; D014544:Uridine Triphosphate